Project Summary Methicillin-resistant Staphylococcus aureus (MRSA) are the most common and lethal causes of infections of hospitalized patients and, over the past decade, have appeared in the community as an increasingly common cause of infections of individuals who both have and do not have healthcare exposure. MRSA are resistant to [unreadable]-lactam (penicillin and derivatives) and many additional antibiotics (multiresistant), severely limiting options for treating serious infections. [unreadable]-lactam antibiotics target the enzymes (penicillin-binding proteins or PBPs) that maintain the integrity of the bacterial cell wall. The gene responsible for methicillin resistance (MR), mecA, encodes a new PBP (PBP2a) that can maintain cell wall structure but is resistant to [unreadable]-lactam inhibition. mecA is carried on a genetic element called a genomic island that inserts into the staphylococcal chromosome at a specific sequence (attB). In addition to housing mecA, this island, called SCCmec, carries genes called ccr that catalyze both insertion and excision of SCCmec. Both the mechanisms of insertion and excision and the epidemiology of various forms of SCCmec suggest that the element is mobile and has moved among staphylococcal strains multiple times in the past thirty years. However, the genetic types of SA that have acquired SCCmec are limited compared to the wide range of strain types of methicillin-susceptible (MS) SA that are available. This proposal seeks to understand how SCCmec is transferred among staphylococci and the specific genetic requirements of recipient MSSA. The three Specific Aims are to 1) Elucidate the molecular mechanisms and target sequences required for ccr-mediated SCCmec insertion and excision;2) Demonstrate that SCCmec can be captured on a conjugative plasmid and transferred among staphylococci by cell-to-cell contact;3) Identify mutations in the genome of the recipient bacterium that must occur for SCCmec and mecA to be stably maintained and to express MR at a clinically relevant level. In addition, since there is epidemiologic and genomic evidence that S. epidermidis (SE), a less virulent staphylococcal species, also carries SCCmec and can serve as a genetic reservoir for this element, we will perform experiments in both SA and SE. The goal of these studies is to identify sequences in SA that can be used as probes to dissect the epidemiology of the spread of MR. In this manner we can trace the history of the emergence and rapid spread of MRSA, prevent future dissemination of SCCmec and support arguments that reduced antibiotic pressure will favor SCCmec loss from host bacteria and lower the environmental prevalence of the MRSA phenotype. In addition, we hope to add to the body of data that associates changes in the bacterial genome with exposure to antimicrobials that target the cell wall and cell membrane. Elucidation of these pathways may provide new targets for chemotherapy and new strategies for treatment.